Method and apparatus for in vivo collection of circulating biological components

ABSTRACT

The invention relates generally to in vivo collection of circulating molecules, tumor cells and other biological markers using a collecting probe. The probe is configured for placement within a living organism for an extended period of time to provide sufficient yield of biological marker for analysis. In some embodiments of the invention, active attraction of biological markers are provided. A partial or complete analytic/detection assembly may also be integrated with the probe.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patent application Ser. No. 10/927,959 filed on Aug. 27, 2004 which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/531,928 filed on Dec. 22, 2003, the disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to devices and methods for collecting and/or detecting biological components in vivo over a period of time. Active methods of collecting biological components are also provided. The detection and/or analysis of the biological components collected by the devices may be performed in vivo or ex vivo.

2. Description of the Related Art

Cancer is one of the leading causes of disease, being responsible for 563,700 deaths in the United States each year (Jemal A et al., Cancer statistics, 2004, CA Cancer J Clin. 2004 January-February; 54(1):8-29). For example, breast cancer is the most common form of malignant disease among women in Western countries and, in the United States, is the most common cause of death among women between 40 and 55 years of age (Forrest A P, Screening and breast cancer incidence, J Natl Cancer Inst. 1990 Oct. 3; 82(19):1525-6.). The incidence of breast cancer is increasing, especially in older women, but the cause of this increase is unknown. Malignant melanoma is another form of cancer whose incidence is increasing at a frightening rate, at least sixfold in the United States since 1945, and is the single most deadly of all skin diseases (Jemal et al., 2004).

One of the most devastating aspects of cancer is the propensity of cells from malignant neoplasms to disseminate from their primary site to distant organs and develop into metastases. The early spread of viable tumor cells is considered a hallmark in cancer progression. Despite advances in surgical treatment of primary neoplasms and aggressive therapies, most cancer patients die as a result of metastatic disease. Animal tests indicate that a substantial frequency of circulating cancer cells from solid tumors establish successful metastatic colonies (Fidler, 1993). Studies have found that the detection of circulating metastatic tumor cells and circulating tumor DNA in the blood of cancer patients correlates with cancer progression. (Hoon D S, et al., Molecular markers in blood as surrogate prognostic indicators of melanoma recurrence, Cancer Res. 2000 Apr. 15; 60(8):2253-7, and Taback B, et al., Circulating DNA microsatellites: molecular determinants of response to biochemotherapy in patients with metastatic melanoma, J. Natl. Cancer Inst. 2004 Jan. 21; 96(2): 152-6, herein incorporated in their entirety by reference)

Thus, the detection of occult cancer cells, DNA and tumor markers in the circulation is important in assessing the level of tumor progression and metastasis. Because subclinical metastasis can remain dormant for many years, traditional surveillance measures such as radiological monitoring with CT scans or MRI and nodal biopsy may lack the sensitivity to detect early disease.

Notwithstanding the foregoing, there remains a need for improved methods and devices for detecting biological components of disease.

SUMMARY OF THE INVENTION

In one embodiment of the invention, a biological surveillance probe for detecting disease is provided. The probe comprises an elongate body having a proximal end and a distal end, an attraction structure attached to the elongate body, wherein the attraction structure is capable of attracting a binding agent. In some embodiments, the attraction structure is a magnetizable structure, a microstructure, a nanotube microstructure, a metallic microporous structure, or a cavity containing a mixture of polymer gel and magnetizable particles. The probe may further comprise a detection assembly. In some embodiments, the detection assembly may be an electrical detection assembly, an impedance-based detection assembly, an ion-exchange membrane detection assembly or a fiberoptic-based assembly. The probe may further comprise a binding agent. In one embodiment, the binding agent comprises an antibody, a fluorescent dye component or quantum dot linked to the binding agent. In one embodiment, the elongate body comprises a stent-like structure. The attraction structure may comprise a magnetizable coating on the elongate body.

In another embodiment, a method for detecting disease is provided. The method comprises the steps of providing a binding agent attraction device, inserting the device into a body, introducing a binding agent into the body, attracting at least a portion of the binding agent to the attraction device, and assessing the binding agent attracted to the attraction device. In some embodiments, the introducing step may be performed by injecting the binding agent into the bloodstream, eluting the binding agent from an implant within the body or ingestion of the binding agent into the body. In some embodiments, the assessing step is performed by impedance-based detection of the binding agent or by optical detection of the binding agent.

In another embodiment of the invention, a method for detecting disease is provided. The method comprises the steps of introducing a binding agent into the body, attracting at least a portion of the binding agent to a location in the body and assessing the attracted binding agent. The assessing step may be performed ex vivo or in vivo. The location in the body may be the position of an attraction device placed within the body. In one embodiment, the binding agent is linked to a fluorescent dye. The assessing step may be performed by assessing the fluorescence of the fluorescent dye levels of the attracted binding agent.

Several embodiments of the present invention provides these advantages, along with others that will be further understood and appreciated by reference to the written disclosure, figures, and claims included herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and operation of the invention will be better understood with the following detailed description of embodiments of the invention, along with the accompanying illustrations, in which:

FIG. 1 is a cross sectional view depicting one embodiment of a probe capable of collecting biological components;

FIG. 2 represents an elevational view of another embodiment of a probe with a guidewire lumen and side port;

FIGS. 3A and 3B are scanning electron micrographs depicting various embodiments of the invention comprising porous structures;

FIGS. 4A through 4D are micrographs illustrating various configurations of the micro-porous tube of a probe;

FIG. 5 is a schematic cross sectional view of an active probe with a polymer gel cavity.

FIG. 6 is a cross sectional view of an active probe with a microporous tip.

FIG. 7 is a cross sectional view of an active probe with a microporous tip and an ion-exchange membrane covering.

FIGS. 8A through 8C are schematic views of one embodiment of a probe and an external activation system. FIG. 8A is a schematic cross sectional view of one embodiment of an external cuff. FIG. 8B is a schematic of one embodiment of a cuff attached to an external unit. FIG. 8C is a schematic view of the probe and external cuff applied to a patient. FIG. 8D is another embodiment of a probe usable with an external activation system.

FIG. 9 is a cross sectional view of an active probe with a fiber optic detection assembly.

FIGS. 10A and 10B depict another embodiment of the active probe with a fiber optic detection assembly.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The detection of occult cancer cells and other biological markers has shown promise in the diagnosis and treatment of disease. For example, the monitoring of patients' blood for circulating tumor cells and other markers may prove advantageous in detecting early tumor progression before metastasis to other organs occurs. Circulating nucleic acids, tumor cells and proteins can be detected in the blood (inclusive of plasma and serum), bone marrow, cavity fluids and cerebrospinal fluid (CSF) of cancer patients which may serve as risk stratification factors, markers for the presence of clinical disease, predictors of subclinical and/or minimal residual disease presence, determinants of treatment response and disease progression, and prognosticators of patient outcome. Other body fluids shown to have the above tumor cells, protein markers, carbohydrate markers or nucleic acids include urine, pleural fluids and peritoneal fluids (ascites). However, assessment of these molecules/tumor cells or components thereof requires a blood sample, which is collected at a single time-point or at multiple time points by deliberate invasion of body tissue (i.e. needle stick).

These methods are often limited by the intermittent and/or low-level presence of cancer cells and markers in the blood. Although new amplification and detection techniques, such as immunochemistry, flow cytometry and reverse transcriptase polymerase chain reaction aid in the detection of early disease markers, these techniques may fail to overcome sampling errors inherent in the blood draws. Because of the constant circulating nature of blood and the limited volume in a particular blood draw, evaluating a single blood sample at one time-point may not accurately represent the quantity and quality of circulating nucleic acids, tumor cells, proteins or other tumor markers for diagnosis, prognosis and monitoring of disease. Sampling error can contribute to the frequent false-negative results found with post-treatment cancer surveillance.

A major problem in detecting tumor cells and tumor markers in blood is that they are not released at any particular time point. Therefore, the probability of detecting the presence of tumor cells or markers may vary or may be unpredictable. In addition, it is known that for certain biological markers, blood flow and release of these markers from tissues are diurnally related and influenced by physical activity of an individual (i.e., climbing stairs). Circulating nucleic acids, tumor cells, proteins etc as described above (here to fore termed circulating molecules or cells or products will be referred as markers or CMC) may also be released transiently into the blood stream by other physiological events and external influences. Repetitive sampling without repetitive invasive procedures would improve the accuracy and sensitivity of detecting molecules circulating in blood.

CMCs appear to circulate in varying levels/concentrations throughout a person's disease course as well as during a single day and or in response to environmental manipulations such as treatment with chemotherapy, hormonal therapy, immunotherapy and radiotherapy as well as with administration of medications. The variations in the stability of these CMCs found in the blood or other body fluids add to the inherent difficulties of an assay that evaluates blood at a single time point. Serial assessment of blood would increase the probability of identifying CMCs and therefore improve their utility as prognostic, predictive and diagnostic assays. However, serial assessments of patients blood requires repeated patient needle sticks which are impractical, inconvenient and uncomfortable to the patient.

A more practical and less intrusive approach would be to introduce a collecting device, probe, biomaterial adhesive matrix, chromatography affinity surface chip or probe, biochip, or particle into the body that would come in direct contact with the blood or body fluid over a period of time. The device may collect, bind or attract the CMCs of interest over time. This product can then be assessed, in vivo or ex vivo, after an interval of elapsed time to provide a more accurate evaluation of those CMCs. One embodiment of the invention comprises a percutaneously inserted device that resides indwelling in the bloodstream and can attract and retain a binding partner such as nucleotides (i.e.: oligos, LNAs (locked nucleic acids), PNAs (peptide nucleic acids), cDNA, nucleic acid probes, chromatographic affinity probes or fragments thereof or their derivatives, complementary fragments or larger) antibodies (i.e.: monoclonal, polyclonal, FAb fragments, etc) proteins or any biological or synthetic material (i.e. biotin-avidin) that is complementary to the CMC in question and that can be assessed in vivo or ex vivo. The desired binding partner(s) are capable of binding the corresponding target marker of interest in a manner in a sufficient concentration and manner that permits retrieval of the probe after an indwelling sample period of time for qualitative or quantitative analysis of the marker.

The ex vivo concept is similar to a “dip stick” approach in assessing a body fluid for a particular molecule. The in vivo concept is a like an implantable physiological monitoring device. A device and approach of such nature will provide a great improvement over current methods of evaluating blood. The evaluation of the CMC can be in the form of conventional monitoring using established in vitro monitoring systems. For example to detect circulating tumor cells or circulating nucleic acids one can use RealTime quantitative PCR and oligonucleotide arrays. For detection of proteins, one can use enzyme-linked immunosorbent assay (ELISA), chromographic affinity assays, etc. For in vivo monitoring it can be through electric or thermal related impulses or direct imaging. Such embodiments are described in further detail below.

One alternative to coating or affixing a binding partner to the surface of a probe is to introduce a mobile binding agent into the body. The mobile binding agent can circulate through the bloodstream or other body compartment while binding to the marker of interest. The mobile binding agent is then concentrated onto a probe using ferromagnetic, electrostatic, electrical, ionic or other type of force. This scheme has the advantage of distributing the binding agent through a larger volume of distribution in the body. This can increase the effective binding rate of the binding agent to the marker, which in turn will improve the yield of the detection process and/or reduce the indwelling time for the probe. The binding agent is then concentrated at a body location for further analysis, under in vivo or ex vivo conditions.

Thus, one embodiment of the invention comprises a method for enhancing the yield of CMC or marker by an attraction and collection probe. The method involves introducing a binding agent into the body that is capable of binding to one or more CMC of interest. The binding agent is typically injected into the body, but diffusion from an eluting component of the probe or a separate eluting implant, ingestion, or transfer into the body transdermally or by suppository may also be possible.

In one embodiment, the binding agent is linked to an attractant that is capable of interacting with an attraction site on a probe to produce an attraction force. The attraction force is capable of causing contact and retention of the binding agent/attractant combination to the attraction site on the probe. In one embodiment, the combination may be attracted to the probe irrespective of whether the binding agent has bound to a CMC or marker.

Distinguishing between the bound and free binding agent collected by the probe may be performed by additional processes. For example, the probe with collected binding agent may be removed from the body and ex vivo separation and analysis of the binding agent may be performed by using processes well known in the art. In another embodiment, the binding agent/attractant combination may undergo a conformation change or activation upon binding to a CMC that may alter the attraction of the combination to the probe.

In another embodiment, the attraction site on the device comprises a microporous structure that attracts the unbound binding agent to a site different or deeper within the microporous structure than cannot be reached by the bound binding agent.

In one embodiment, a time period is provided for the binding agent to bind at least a portion of the CMC in the volume and compartment of distribution. The implantation time of the attraction probe may be shorter or longer than the time period provided for the binding agent to bind the CMC. This time period may be selected based upon the binding kinetics between the binding agent and CMC, the kinetics between the CMC and/or binding agent with respect to body tissues and compartments, the anticipated CMC levels in the body, the method of binding agent introduction into the body, and other factors known to those skilled in the art. Once bound to the CMC, a probe within the body can be activated to attract the bound CMC to the probe. Activation of a probe is described in greater detail below. In another embodiment, a probe that is inherently magnetic and does not require activation may be used to attract the complexes. In another embodiment of the invention, yield of CMC may be enhanced by reducing interference from substances or components that can affect the binding of the CMC. In one embodiment, an ionically charged binding surface provides a repelling force against certain classes of interfering materials. In another embodiment, a filter is provided between the body environment and the binding surface of the probe. The filter is capable of reducing passage of material through the filter based upon one or more characteristics. These characteristics include but are not limited to particle size, particle charge, etc. Embodiments for enhancing the yield of CMC are provided in greater detail below.

The detection time of the probe may be continuous, over multiple intervals, or event-driven. Inactivating the detection mechanism at other times may reduce fibrin deposition and other deleterious processes during periods of low yield. For example, increased core body temperature or increased serum potassium levels are correlated with cell lysis of certain cancers and detection during of these events may enhance the yield of interval collection and detection schemes. Other event-based detection periods may include time period to assess a patient's response to therapy through detection of components related to cellular death. This allows measurement of a patient's response, for example, to chemotherapy and/or radiation therapy, which can then be optimized to for treatment effect or to minimize side effects.

This device(s) can be inserted surgically, percutaneously or intravenously into the blood stream, peritoneal cavity or bone marrow such that continuous contact with circulating blood, and/or body fluids is ensured. The product can then be collected for analysis in a routine fashion or monitored over time. Several indwelling devices are currently available that coexist with the patient that in long-term contact with the blood and patients body fluids without inducing an adverse reaction. These devices also do not significantly impair everyday patient activities of daily living. These devices include but are not limited to centrally or peripherally inserted intravenous catheters, pacemakers and their leads, automatic internal converter defibrillators, hemodialysis catheters, peritoneal catheters and prosthetic grafts.

One example of the proposed device is a coated catheter, guidewire or filament, chip, biomaterial and/or matrix that can be inserted through a centrally or peripherally placed intravenous catheter or implantable catheter/material into body fluids such as peritoneal cavity, bone marrow, cerebrospinal fluid, etc. This device can then dwell in continuous or intermittent contact with the bloodstream and/or body fluids to improve yield of collecting tumor cells and components thereof, circulating nucleic acids, and proteins, or other items previously mentioned and/or for prolonged or continuous in vivo or ex vivo monitoring of marker presence or activity. Monitoring time can vary in vivo from one to several days to weeks or longer. This may provide valuable information on markers of subclinical and/or minimal residual cancer presence and determinants of treatment response and disease progression. Such devices may also be used to monitor host states for other disease progression patterns, including but not limited to infectious processes and organ transplant rejection.

The invention described allows for continuous invasive monitoring of CMCs. Through a percutaneous approach, a catheter can be placed into the vasculature of a patient for continuous monitoring of circulating tumor cells and/or their component. Monitoring of CMCs may have diagnostic and prognostic value in patient care as well as serve as an improved mechanism for monitoring response to treatment. This indwelling catheter, for example, may attract one or more complementary substrates which can include but are not limited to RNA, DNA, oligonucleotides, proteins, carbohydrates, antibodies, LNAs, PNAs, probes, or any component thereof and/or aforementioned in this application that has affinity for binding to the CMC. When the desired substrate is attracted to the catheter, chip or any device mentioned in this context contained therein, the substrate can be quantified and evaluated for information that can be conveyed to a self-embedded or external detector. In addition, this catheter or device (including nanoparticles, nanodevices, microfabricated devices, etc) and/or with an associated chip or other device containing complementary substrate to the source(s) for identification to which contains the attracted substrate of interest can be removed for ex vivo analysis whereby the information obtained would provide both qualitative and quantitative data.

In addition to enhancing the sensitivity of detecting cancer and cancer recurrence, the invention allows assessment of circulating tumor cells and also would provide a rapid monitoring system to determine if a specific therapy is effective.

In one embodiment of the invention, continuous surveillance/monitoring of circulating nucleic acids (including RNA, double stranded and single stranded DNA, chimeric RNA/DNA), tumor cells, fetal cells, transplant allogeneic cells, transfected cells, proteins, infectious disease nucleic acids, proteins, carbohydrates (including glucoproteins, gangliosides and phospholipids) in any complete components or fragment forms, is performed to assess the presence and/or progression of disease. These molecules will be detected in serum, plasma, whole blood, bone marrow, CSF, lymphatic fluid, pleural or peritoneal fluids, urine or other body fluids in patients with cancer, hyperplasia, pregnancy (including prenatal diagnosis), patients with infectious diseases symptomatic or asymptomatic with other medical conditions such as infectious disease, autoimmune diseases, inflammatory diseases, cardiovascular disease (including myocardial infarction, unstable angina and congestive heart failure), neurovascular diseases (e.g., ischemic events, stroke, anemia), pulmonary disease (including acute respiratory distress syndromes, fibrosis, pulmonary hypertension, emphysema, asthma, chronic obstructive pulmonary disease), renal disease (infection, hypertension nephropathies, nephritis, renal insufficiency and renal failure), trauma patients, organ failure, critical care patients, and transplant patients (including allogeneic and xenogeneic).

A. Binding Partners

The terms “binding partner”, “binding agent” or “member of a binding pair” refer to molecules that specifically bind other molecules (e.g., a marker of interest) to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc. In certain embodiments, the binding is predominantly mediated by noncovalent (e.g. ionic, hydrophobic, etc.) interactions.

One or more binding partners that specifically bind a target marker to be detected are attracted to the attraction structure on the probe of the invention. The binding partner(s) used in this invention are selected based upon the target marker(s) that are to be identified/quantified. Thus, for example, where the target marker is a nucleic acid the binding partner is preferably a nucleic acid or a nucleic acid binding protein. Where the target marker is a protein, the binding partner is preferably a receptor, a ligand, or an antibody that specifically binds that protein. Where the target marker is a sugar or glycoprotein, the binding partner is preferably a lectin, and so forth. A device of the invention can involve several different types of binding partners, for example, multiple nucleic acids of different sequence and/or nucleic acids combined with proteins in the same device. The latter would facilitate, e.g., simultaneous monitoring of gene expression at the mRNA and protein levels. Other combinations of different types of binding partners can be envisioned by those of skill in the art and are within the scope of the invention. Furthermore, the binding partner may be combined with an optically sensitive dye to facilitate assessment of bound CMCs.

Methods of synthesizing or isolating such binding partners are well known to those of skill in the art. For example, nucleic acids for use as binding partners in this invention can be produced or isolated according to any of a number of methods well known to those of skill in the art. In one embodiment, the nucleic acid can be an isolated naturally occurring nucleic acid (e.g., genomic and/or mitochondrial DNA, cDNA, mRNA, etc.). Methods of isolating naturally occurring nucleic acids are well known to those of skill in the art (see, e.g., Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

1. Antibody-based

Antibodies or antibody fragments for use as binding partners can be produced by a number of methods well known to those of skill in the art (see, e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, and Asai (1993) Methods in Cell Biology Vol. 37. Antibodies in Cell Biology, Academic Press, Inc. N.Y.). In one embodiment, antibodies are produced by immunizing an animal (e.g., a rabbit) with an immunogen containing the epitope to be detected. A number of immunogens may be used to produce specifically reactive antibodies. Recombinant proteins are the preferred immunogens for the production of the corresponding antibodies. The antibodies may be monoclonal or polyclonal. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides are also suitable and can be made using standard peptide synthesis chemistry (see, e.g., Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.) Preferably, human or humanized antibodies are used to prevent host anti-xenogen antibody production. These antibodies may include antibodies derived from hybridomas (tumor cells fused with antibody-producing mammalian cells), humanized chimerics, Epstein-Barr Virus transformed B-cells and transgenic antibodies.

Methods for producing polyclonal antibodies are also well known to those of skill in the art. In one embodiment, an immunogen is mixed with an adjuvant and an animal is immunized. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the immunogen. When sufficient titers of antibody to the immunogen are obtained, blood is collected from the animal and an antiserum is prepared. If desired, the antiserum can be further fractionated to enrich for antibodies having the desired reactivity. The animal may be a monoclonal mouse, rat, rabbit, chicken or other animal known in the art.

Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. In one embodiment, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (See, Kohler and Milstein (1976) Eur. J. Immunol. 6: 511-519). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yields of the monoclonal antibodies produced by such cells can be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, DNA sequences encoding a monoclonal antibody or a binding fragment thereof can be isolated by screening a DNA library from human B cells according to the general protocol outlined by Huse et al. (1989) Science, 246:1275-1281. Such sequences can then be expressed recombinantly.

In one embodiment of the invention, the technique comprises injection of an antibody or fragment of an antibody (referred to as Ab) into a patient or organism and to use a device that can allow capture of protein, circulating tumor cells, DNA or RNA in the blood stream or body cavity. The device will be able to attract Ab in high density. The Ab may be natural, recombinant (chimeric, Fab, scFv, etc.), or genetically engineered. Preferably the Ab will be human to prevent anti-foreign antibody responses (i.e. human antibody response to mouse antibodies; HAMA). The device can be removed after insertion into the blood stream to be monitored for biomarkers or cells it can attract and capture. The insertion device can be a catheter, array chip, capture vessel, capture filter, entrapment or attraction device. The device can be inserted for 1, 2, 3, 4 . . . 24 hrs or days or weeks. Monitoring of the captured biomarker or cells may be assessed in vivo or ex vivo utilizing known techniques depending on the biomarker or cell type. The biomarker or cells captured can be assessed quantitatively or qualitatively. In another approach the biomarker or cells captured will be monitored in vivo utilizing a signaling indicator based on electrical, colorimetric or activation signals.

In attracting cells to the device, specific Ab that bind to cell surface markers of cancer cells may be used. Cancer cells have distinct markers on their cell surface that distinguish them from normal cells. This has been demonstrated by immunohistochemistry (Racila E et al., Detection and characterization of carcinoma cells in the blood, Proc Natl Acad Sci U S A. 1998 Apr. 14;95(8):4589-94). These antibodies can be used to target epithelial origin cells, tumor cells originated from specific tissues, non-epithelial origin cells (i.e. melanoma). Circulating tumor cells are found in the blood stream and body fluids of cancer patients (Hoon D S, et al., “Detection of occult melanoma cells in blood with multiple-marker polymerase chain reaction assay” J Clin Onc. 1995 August; 13(8); 2109-16, and Hoon D S, et al., “Molecular markers in blood as surrogate prognostic indicators of melanoma recurrence” Cancer Res. 2000 Apr. 15; 60(8): 2253-7.). Tumor cells spread to distant organs via the blood stream, lymphatic ducts or body fluids or body cavities. The spread of tumor cells can eventually lead to tumor growth at distant sites from the original tumor, thus producing metastasis. Growth of metatastatic tumor sites can lead to death.

Detection of tumor cells can be used as an indicator of disease spread, tumor aggressiveness, potential to spread to other organs, and presence of disease in individuals who are otherwise diagnosed as disease-free by conventional means. Detection of tumor cells in vivo may be advantageous in some circumstances over ex vivo detection. The approach will allow better capture of early disease. One cannot predict disease spreading or volume through single blood draw of a small amount of blood or body fluid. One approach comprises catching tumor cells through having an attraction and capturing system in the blood stream or body fluid to access a larger vascular or fluid volume and/or for a longer period of time to increase the yield of marker recovered. This is may be advantageous when capturing occult circulating metastatic or leukemic tumor cells. The cell surface marker can be a protein, glycoprotein, glycolipid, peptide epitope, conformational biological epitope or multiple disease or tumor markers. The device may have more than one Ab attached to it to improve sensitivity and capturing ability. The Ab may be to multiple epitope sites of a single biomarker antigen. The tumor cells captured may be dislodged when the device is removed and assessed by the following ex vivo methods: immuno-histochemistry, DNA, mRNA and/or proteomics. In other embodiments, the binding complexes may remain on the probe and assessed in situ.

The isolation of the cells may involve physical removal, direct solvent removal specific to that biomarker's physical-chemical properties or cessation of the active attraction force of the probe to release the binding agent/biomarker complex. For example DNA and RNA from tumor cells can be extracted directly from the tumor cells after isolation. Isolation of DNA or RNA can be by solvents used for nucleic acids. This can be accomplished directly or after the cells have been dislodged. RNA and DNA can be detected by hybridization to a specific probe, polymerase chain reaction (PCR) or related monitoring approach. The assessment of nucleic acids from the tumor cells can provide quantitative and qualitative analysis. Even if non tumor cells are captured, the specificity of the analysis can be optionally increased through a second tier analysis. Sensitivity of the analysis can be further be enhanced through amplification of the nucleic acids by PCR or related methods, incorporating specific probes or detection systems ex vivo. Specificity and sensitivity ex vivo for the specific nucleic marker can be approached using current technologies. The DNA markers may comprise microsatellites, mutations, translocations, insertions, amplifications, SNPs or chromatin/DNA complexes. The RNA markers can comprise specific genes in whole or part in the form of mRNA.

Protein, glycoprotein, or glycolipid analysis can be detected by antibody, mass spectrophotometry, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS), affinity assay, chromatographic approach. The approach can be directly from the device or removal of the biomarker by some solvent, physical method or reagent to a vessel where it can be processed. The detection can be in the form of an affinity matrix chip for the specific biomarker type.

The Ab used with the device can be a natural antibody produced by human or some animal B cells in the form of polyclonal or monoclonal antibody. The Ab can be a recombinant antibody that is released from transfected mammalian or prokaryotic cells. The Ab can be a fragment of an antibody such as scFV, FV or Fab fragment that has specific recognition of the biomarker or cell epitope. The Ab can be a genetically engineered Ab that has a specific attachment moiety or detection ability.

The Ab used with the device can be polyclonal or monoclonal antibody to a specific epitope or multiple epitopes to a specific biomarker or epitope. It can consist of multiple Ab to multiple biomarkers. The latter will allow higher sensitivity and capturing ability.

2. Protein-based

In one embodiment, the binding partner is a binding protein. Suitable binding proteins include, but are not limited to, receptors (e.g., cell surface receptors), receptor ligands (e.g., cytokines, growth factors, etc.), transcription factors and other nucleic acid binding proteins, as well as members of binding pairs, such as biotin-avidin.

Binding proteins useful in the invention can be isolated from natural sources, mutagenized from isolated proteins, or synthesized de novo. Means of isolating naturally occurring proteins are well known to those of skill in the art. Such methods include, but are not limited to, conventional protein purification methods including ammonium sulfate precipitation, affinity chromatography, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y.). Where the protein binds a target reversibly, affinity columns bearing the target can be used to affinity purify the protein. Alternatively the protein can be recombinantly expressed with a HIS-Tag and purified using Ni.sup.2+/NTA chromatography.

In another embodiment, the binding protein can be chemically synthesized using standard chemical peptide synthesis techniques. Where the desired subsequences are relatively short, the molecule may be synthesized as a single contiguous polypeptide. Where larger molecules are desired, subsequences can be synthesized separately (in one or more units) and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond. This is typically accomplished using the same chemistry (e.g., Fmoc, Tboc) used to couple single amino acids in commercial peptide synthesizers.

The technique will involve binding of free circulating proteins, peptides or protein complexes via an affinity matrix or antibody or ligand (referred to as affinity substrate; AS) that is then attracted to a device and allows capture of proteins, peptides or glycoproteins from the blood stream or body cavity. The device can attract the bound AS in high density. The device can be removed after insertion into the blood stream to be analyzed for biomarkers it can capture. The insertion device can be a catheter, array chip, capture vessel, capture filter, entrapment device. The device can be inserted for 1, 2, 3, 4 . . . 24 hrs or days or weeks. Monitoring of the captured biomarker or cells will be assessed ex vivo utilizing known techniques depending on the biomarker type. The biomarker captured can be assessed quantitatively or qualitatively. In another approach the biomarker captured will be monitored in vivo utilizing a signaling indicator based on electrical, colorimetric or activation signals.

Protein and glycoprotein analysis can be detected ex vivo by antibody, mass spectrophotometry, affinity assay, chromatographic approach. The approach can be directly from the device or removal of the biomarker by some solvent, physical method or reagent to a vessel where it can be processed.

The antibody used with the device can be a natural antibody produced by human or some animal B cells in the form of polyclonal or monoclonal antibody. The antibody can be a recombinant antibody that is released from transfected mammalian or prokaryotic cells. The antibody can be a fragment of an antibody such as scFV, FV or FAb fragment that has specific recognition of the biomarker or cell epitope. The antibody can be a genetically engineered antibody that has a specific attachment moiety or detection ability.

The AS can be in the form of affinity matrix material that is specific or non-specific for particular protein properties. The former is preferable. For non-specific affinity matrix materials (not to a specific biomarker), the AS can be based on charge to attract hydrophilic or hydrophobic molecules. The antibody or ligand substrate attracted by the device can be directed towards a specific epitope or multiple epitopes to a specific biomarker. It can comprise multiple AS to multiple biomarkers. The latter will allow higher sensitivity and capturing ability.

Optionally, a chip-based detection system may be used. For example, DNA/oligonucleotide chip detection involves attachment or incorporation of a chip into a device to be inserted into the blood stream or body cavity. The assessment of components attracted to the chip may be performed in vivo directly through electronic or chemical signaling or ex vivo by a detection device. For DNA analysis this will include microsatellite analysis for loss of heterozygosity (LOH) or by single nucleotide polymorphism (SNP). Other genomic DNA markers can include mutations, amplifications and translocations. The analysis may involve specific or multiple sites of the chromosomal or mitochondrial DNA from tumor cells. RNA analysis will involve assessment of mRNA of transcripts of specific genes related to the tumor cells. The mRNA transcript may be of the whole or part of the full transcript or a truncated derivative of the transcript. The procedure may also include chromatin and DNA complexes (histone proteins) related to specific genomic regions of tumor cells. The procedure may encompass assessing acetylation and deacetylation of chromatin regions of specific genomic regions, methylated or non-methylated. The procedure may encompass assessing methylated or non-methylated regions of the genomic regions such as promoter related-regions of tumor-related genes. The chip may be inserted for 30 min, 1, 2, 3 . . . 24 hr and removed for assessment or assessed directly.

B. Attraction of Binding Partner to Probe

As previously mentioned, the desired binding partner(s) are attracted to the attraction structure or site on the probe in a sufficient concentration and manner to be capable of binding the corresponding target marker of interest. The attraction is performed in a manner that permits retrieval of the probe after an indwelling sample period of time for qualitative or quantitative analysis of the marker. In one embodiment, the linkage between the binding partner and the attraction or substrate surface on or attached to the probe is magnetic, but other attraction mechanisms may also be used. Various embodiments of an attraction system are provided in greater detail further below.

C. General Probe Configurations

Referring to FIG. 1, there is disclosed a CMC or marker binding and retrieval probe 10 in accordance with one aspect of the present invention. Although the probe 10 will be described primarily in terms of an insert to be temporarily placed down an existing access port or sheath into the cardiovascular system, for retrieving a marker from blood, the present inventors contemplate broader applicability as will be apparent to those of skill in the art in view of the disclosure herein. Existing access ports or sheaths include but are not limited to Hickman catheters, Portacath catheters, peripherally inserted central catheter (PICC) lines, femoral, jugular, or subclavian central venous lines, radial arterial catheters and peripheral venous lines. Furthermore, procedures such as transseptal puncture and transjugular intrahepatic puncture, may be used to access other body sites such as the arterial chambers of the heart or the portal vein, respectively.

For example, the probe may be adapted for direct access to a target site, without the use of a distinct tubular access catheter. In general, whether used with an access sheath or as a stand alone device, the dimensions of the probe can be optimized by persons of skill in the art in view of the present disclosure to suit any of a wide variety of target sites. For example, the probe of the present invention can be used to obtain samples from large and small arteries and veins throughout the cardiovascular system, as well as other lumens, potential spaces, hollow organs and surgically created pathways. Marker collection (tumor and/or non-tumor) may be accomplished in blood vessels, body lumens or cavities, such as the lymphatic system, esophagus, trachea, urethra, ureters, fallopian tubes, intestines, colon, biliary ducts, spinal canal and any other locations accessible by a flexible or rigid probe which may contain a specific binding partner of diagnostic value. The probe 10 may also be adapted for direct advance through solid tissue, such as soft tissue or through bone, for site specific monitoring of a binding partner of interest.

The probe 10 generally comprises an elongate body 16 extending between a proximal end 12 and a distal functional end 14. The length of the body 16 depends upon the desired access site and the desired placement site for the distal end 14. For example, lengths in the area of about 1 cm to about 20 or about 30 cm may be useful in applications that require the catheter to be advanced down a relatively short tubular access sheath. Longer lengths may be used as desired, such as on the order of from about 120 cm to about 140 cm for use in percutaneous access at the femoral artery for placement of the distal end 14 in the vicinity of the coronary artery. Intracranial applications may call for a different catheter shaft length depending upon the vascular access site, as will be apparent to those of skill in the art.

Many markers of interest, however, may be equally retrievable at any point throughout the cardiovascular system, in which case the probe 10 may be adapted to advance down any convenient access port that may have been placed for other diagnostic or therapeutic use. Devices in accordance with the present invention may also be adapted for exposure to blood by coupling to any of a variety of ports on extracorporeal circulation systems as will be apparent to those of skill in the art in view of the disclosure herein.

In the illustrated embodiment, the body 16 is divided into at least a proximal section 33 and a distal binding zone 34. In general, distal binding zone 34 is adapted to carry a attraction structure for the marker of interest, as discussed previously, and may or may not be otherwise structurally distinct from the proximal section 33.

At least a portion of the proximal section 33 of body 16 may be produced in accordance with any of a variety of known techniques for manufacturing catheter bodies, depending upon the desired clinical performance. For example, the body 16 may be formed by extrusion of any of a variety of appropriate biocompatible polymeric materials. Known materials for this application include high density polyethylene, polytetrafluoroethylene, nylons, PEEK, PEBAX and a variety of others such as those disclosed in U.S. Pat. No. 5,499,973 to Saab, the disclosure of which is incorporated in its entirety herein by reference. Alternatively, at least a proximal portion or all of the length of body 16 may comprise a spring coil, solid walled hypodermic needle tubing, or braided reinforced wall, as is understood in the catheter and guidewire arts. Whether metal or polymeric or a hybrid, the body 16 may be hollow or solid depending upon the nature of the binding system and other desired capabilities.

In one cardiovascular example, the body 16 is provided with an approximately circular cross-sectional configuration having an external diameter within the range of from about 0.025 inches to about 0.100 inches. In accordance with one embodiment of the invention, the body 16 has an average external diameter of about 0.042 inches (4.2 f) throughout most of its length. Alternatively, generally rectangular, oval or triangular cross-sectional configurations can also be used, as well as other noncircular configurations, depending upon the method of manufacture, desired surface area, flexibility, access pathway and other design considerations that may be relevant for a particular application.

Dimensions outside of the ranges identified above may also be used, provided that the functional consequences of the dimensions are acceptable for the intended purpose of the catheter. For example, the lower limit of the cross section for any portion of body 16 in a given application will be a function of the number of fluid or other functional lumens, if any, contained in the probe, together with the desired surface area to be available for the binding partner, as will be discussed.

Probe body 16 should also have sufficient structural integrity (e.g., column strength or “pushability”) to permit the probe to be advanced to a desired target site without buckling or undesirable bending.

The proximal end 12 of the probe 10 may be provided with a grip 46 such as a polymeric cap 48 which may be molded or otherwise secured to the proximal end 12 of the body 16. Preferably, the cap is provided with a complementary surface structure to allow a removable connection between the cap and the proximal end of an intravenous catheter or other device through which the probe 10 will achieve contact with blood or other body fluid. Removable attachment may be accomplished by using any of a wide variety of clips, twist fasteners such as Luer connectors, interlocking snapfit connectors, or friction fit connectors as will be appreciated by those of skill in the art in view of the disclosure herein.

The axial length of the probe 10 is preferably precisely calibrated to match the particular access catheter with which it is to be used, to provide a reproducible length of the binding zone to be exposed to the sample of interest.

Referring to FIG. 2, there is disclosed another embodiment of probe 10. The proximal end 12 of probe 10 is provided with a manifold 18 having one or more access ports as is known in the art. Manifold 18 may be provided with a guidewire port 20 in an embodiment where over-the-wire navigation of the probe may be desired. An infusion port 22 may be provided with or without the guidewire port. The infusion port is in fluid communication with the binding zone through an infusion lumen. This allows periodic or continuous infusion of saline, heparin or other media to prevent “clogging” or coating of the binding zone over time, by natural clotting or other processes which may interfere with the efficacy of the binding chemistry. Additional access ports may be provided as needed, depending upon the desired capabilities of the catheter. Manifold 18 may be injection molded from medical grade plastics or formed in accordance with other techniques known in the art.

The distal end 14 of the probe 10 may be provided with an atraumatic distal tip 25 which may include a guidewire exit port 26 in a guidewire lumen embodiment as is known in the art. A radiopaque marker (not illustrated) may be provided on the probe body 16 in the case of relatively long probes to facilitate positioning of the probe as is known in the art. Suitable marker bands can be produced from a variety of materials, including platinum, gold, and tungsten/rhenium alloy.

The distal zone of the probe is provided with an attraction structure, capable of attracting a marker of interest. As used herein, the term marker refers to any CMC discussed above, as well as any other cell, cell fragment, protein, peptide, glycoprotein, lipid, glycolipid, proteolipid, or other molecular or biological material that is uniquely expressed (e.g. as a cell surface or secreted protein) by diseased cells, or is expressed at a statistically significant, measurably increased or decreased level by diseased cells, or in association with a disease state of interest (e.g. a protein expressed by an infectious agent associated with disease), or is expressed at a statistically significant, measurably increased or decreased level by diseased cells compared to normal cells, or which is expressed by non-diseased cells in association with disease (e.g. in response to the presence of diseased cells or substances produced therefrom). Disease markers can also include specific DNA or RNA sequences marking a deleterious genetic change, conformational change compared to baseline or normal, or an alteration in patterns or levels of gene expression significantly associated with disease. Disease markers include breast cancer markers.

The term cancer marker refers to a subset of disease markers, namely any protein, peptide, glycoprotein (including but not limited to mucins, mucoid and amyloid glycoproteins), lipid, glycolipid, proteolipid, or other molecular or biological material that is uniquely expressed (e.g. as a cell surface or secreted protein) by cancerous cells, or is expressed at a statistically significant, measurably increased or decreased level by cancerous cells compared to normal cells, or which is expressed by non-cancerous cells in association with cancer (e.g. in response to the presence of cancerous cells or substances produced therefrom). Cancer markers can also include specific DNA or RNA sequences marking a deleterious genetic change, conformational change, or an alteration in patterns or levels of gene expression significantly associated with cancer.

a. Attraction Structure

The attraction structure may be configured with an increased surface area to provide an increased number of interaction sites on the probe. The surface area may be increased by providing an increased longitudinal length, increased diameter or cross-section through at least a portion of the distal zone. In addition, or alternatively, at least a portion of the distal zone may comprise a porous material and/or microstructure to increase the surface area. Non-limiting examples of porous materials include porous polymers, ePTFE, PTFE, polyurethane, silicone, foam, or a ceramic with a porous surface (e.g., titanium nitride, titanium carbide, carbon, and silicon carbide). Various techniques for depositing material on the probe surface to provide a porous structure may also be used and include ion beam deposition, sintering, sputtering, ion implantation, laser surface alloying, electroplating, physical or chemical vapor deposition, chemical or physical etching, grit blasting, plasma and thermal spray coating. Other materials that can be applied to the probe surface include iridium oxide, graphite and platinum black. The surface area may be increased through microstructures on the binding zone surface, formed from processes including but not limited to mechanical roughening of the probe surface, laser drilling or metal sintering onto the probe. The probe may also be manufactured using microporous tubing, porous fabric or and polymers, or carbon fiber bundles, and nanotubes. The surface area of the binding zone may be configured by one skilled in the art depending upon the expected release pattern, degradation and metabolization pathways and binding kinetics of the CMCs of interest.

FIGS. 3A and 3B represent scanning electron micrographs (SEM) of various porous configurations that provide an increased surface area for the probe. FIG. 3A depicts one embodiment of the invention comprising a microporous zone formed by vapor deposition. FIG. 3B depicts another embodiment of the invention formed with sintered metal beads. One skilled in the art will understand that a variety of metals may by used for a sintered porous surface, including but not limited to platinum, platinum/iridium and other platinum group metals or alloys thererof, titanium, titanium alloys and 316L stainless steel. In one embodiment, the sintered metal zone has an average pore size of about 5 microns to about 150 microns to allow particle access into the microporous structure. In other embodiments, an average pore size of about 5 microns to about 100 microns may be used. In one example, a sintered metal porous zone has an average pore size of about 10 microns to about 50 microns. Microporous structures will typically have a porosity between about 10% to about 80%. In some embodiments, the porous layer has a porosity of about 10% to about 60%, and preferably about 40%.

Other binding zone structures that increase the surface area are shown in FIGS. 4A through 4D. FIG. 4A is a photograph of a porous fabric. FIG. 4B depicts a porous polymer. FIG. 4C depicts laser drilled holes in a polymer surface and FIG. 4D depicts a nanotube microstructure for providing an increased surface area.

b. Magnetic attraction

One embodiment of the invention utilizes placement of an indwelling metallic device into the circulatory system or other body organ which can conduct electromagnetic current. The device may be regulated and monitored from an ex vivo source. Intravenous injection of magnetic beads or particles coupled to complementary nucleotides (i.e.: oligos, CpG motifs, LMAs, peptide nucleic acids (PNAs), cDNA, probes, nucleic acid sequences or fragments thereof or their derivatives, complementary fragments or larger) antibodies (i.e.: monoclonal, polyclonal, Fab fragments etc) proteins (i.e.: albumin, prealbumin) or any biological or synthetic material (i.e. biotin-avidin). These complementary binding partners can bind circulating tumor cells or any disease-associated components thereof that may be in body. The indwelling venous catheter is induced with electromagnetic current to bring the magnetic particle complexed to the CMC of interest in contact with the indwelling/inserted catheter monitoring device. In one embodiment, a second antibody to that is complementary to the tumor cell and or its components which carries a fluorescent label would be brought into the vicinity of the catheter by its complementary binding to the substrate, bringing the fluorescent molecule in proximity to the catheter which could optically detect the fluorescence and convert this to a quantitative readout that corresponds to the amount of circulating tumor cells or its components present. These and other embodiments of the invention are described in greater detail below.

FIG. 5 depicts one embodiment of the invention comprising a method of detecting CMC bound to a collection probe. The collection probe 100 comprises an elongate body 102 have a proximal end 104 and a distal end 106. A cavity 108 containing a polymer gel 110 is located on the elongate body 102. The cavity 108 is typically located at the distal end 106 of the elongate body 102, but may also be located at other positions along the length of the probe 100. The probe 100 may have more than one cavity. The cavity 108 may have any of a variety of shapes sufficient to hold a volume of polymer gel, including but not limited to a spherical, box-like, cylindrical, conical or frusta-conical shape. The cavity 108 may have an axial cross sectional shape, as measured with respect to the longitudinal axis of the probe, of about 0.4 mm² to about 5 mm², and preferably about 1 mm² to about 2 mm². The cavity 108 may have a longitudinal length of about 2 mm to about 10 mm, and preferably about 2 mm to about 3 mm. The material defining the cavity 108 and elongate body 102 may be any of a variety of materials used in the art for catheter bodies, including but not limited to high density polyethylene, polytetrafluoroethylene, nylons, PEEK, PEBAX and a variety of others such as those disclosed in U.S. Pat. No. 5,499,973 to Saab, the disclosure of which is incorporated in its entirety herein by reference. In some embodiments of the invention, the materials used have electrically insulative properties. In some embodiments, the materials used have a hardness of about 30 A to about 60 A, preferably about 20 A to about 40 A. The polymer gel 110 may comprise silicone, polyurethane, hydrogel, PLA or any other porous polymer gel known in the art. The polymer gel is mixed with any of a variety of conductive particles, including but not limited to carbon, metal or a metallic metal. The distal ends 112 of two or more lead wires 114, 116 are in contact with the polymer gel 110 in the cavity 108 and run along the length of the elongate body 102 of the probe 100 and terminate at the proximal end 104 of the elongate body 102 at one or more electrical connectors 118. The lead wires 114, 116 will typically comprise copper or Monel electrical wire with a diameter of about 30 AWG to about 50 AWG. One skilled in the art will understand that any of a variety of electrical wires may be used for the invention. The wire may optionally have a Teflon or polyimide insulation coating, generally about 10 micron to about 100 micron in average thickness. The electrical connector is configured to attach to an electrical current generator and current measuring system 120. The electrical connector 118 may be a standard connector as known by those with skill in the art, or a proprietary connector. The electrical connector 118 may also be of a high-impedance and/or high-leakage isolation type of connector. In other embodiments, the electrical current generator and current measuring system 120 is directly connected to the lead wires 114, 116 and an electrical connector 118 is not required. The electrical current generator and current measuring system 120 may be configured to measure electrical resistance in the range of about 10 Ohms to about 100K Ohms and run on a AC or DC voltage system of about 2V to about 50V. One skilled in the art may select and/or configure the electrical current generator and current measuring system 120 to the particular embodiment of the invention.

To use one embodiment of the invention, the patient is injected with a magnetically labeled antibody with at least some specificity for the CMC of interest. The magnetic label of the antibody comprises iron or ferrite based beads with about a 2 micron to about a 10 micron particle size. The impedance-based probe is inserted into the circulatory system of the patient and the electrical current generator and current measuring system is activated. The electric circuit generates a magnetic field within the polymer gel of the probe that is capable of attracting the magnetically linked antibody or binding partner. In some embodiments, the magnetically linked antibody or binding partner lodges in the polymer gel as they are attracted to the magnetic field. In other embodiments, the magnetically linked binding partner remains in contact with the polymer gel primarily by the magnetic field and may release back into the body circulation if the magnetic field is shut off. In some embodiments, the measurement of the electrical resistance can be used to determine the duration that the probe is left in the patient and/or electrical generator is activated. As the bound or unbound antibodies are attracted, the changes in the electrical resistance as measured through the gel may indicate when all or a sufficient amount of antibody has been collected. The probe is removed from the patient. The antibody bound to a CMC of interest is separated from the probe or differentiated from the unbound antibody and analyzed. Separating and/or distinguishing a bound binding partner from an unbound binding partner is well known in the art. In some embodiments of the invention, the unbound antibody is separated from the probe while the CMC-bound antibody remains on the probe. An impedance measurement system may then be used to analyze the presence of remaining bound antibody on the probe. Impedance based detection of biological product is further described by Lee et al in U.S. Publication No. 2004/0100284A1, herein incorporated in its entirety by reference.

Referring to FIG. 6, in another embodiment of the invention, a magnetizable microporous structure 122 is provided rather than a cavity filled with polymer gel. The lead wires 114, 116 of the probe 100 contact the microporous structure 122 and are capable of creating a magnetic field using the microporous structure 122. The lead wires 114, 116 may also be integral with the microporous structure 122. The microporous structure typically comprises one or more metals such as platinum, or platinum/iridium (90/10% to about 80/20%), 316L stainless steel or titanium (CP grade 1 to 4). One skilled in the art will understand that other magnetizable microporous structures may be used. The microporous structure 122 will typically have a cross-sectional area of about 0.25 mm² to about 5 mm², and preferably about 1 mm² to about 2 mm². The cavity may have a longitudinal length of about 1 mm to about 10 mm, and preferably about 2 mm to about 3 mm. The microporous structure 122 will typically have a porosity of about 10% to about 60%, and preferably about 40%, with a particle size ranging from about 5 microns to about 100 microns. The use of the magnetizable microporous probe is similar to that described previously for the polymer gel embodiment of the invention. During magnetic attraction of the magnetically labeled antibody, the bound and unbound antibody may or may lodge within the microporous structure of the probe.

In another embodiment of the invention, depicted in FIG. 7, the microporous structure 122 is covered with an ion-exchange membrane 124 that is selectively permeable based upon one or more characteristics, including but not limited to particle size and ionic charge. In one embodiment, the membrane 124 has a thickness of about 20 microns to about 150 microns, and a pore size of about 40 microns to about 100 microns. One skilled in the art can select a particular membrane configuration based upon the desired filtering characteristics. For example, Nafion (DuPont, Del.) is a synthetic polymer membrane with ionic properties that can be used to provide relative increased permeability to circulating tumor cells of cDNA.

In another embodiment of the invention, illustrated in FIGS. 8A through 8C, the probe 100 comprises a magnetizable attraction site 123 which can generate a magnetic field by applying one or more other magnetic fields about the site 123. The other magnetic fields may originate from magnets external to the patient's body that are positioned adjacent to the probe 100. Referring to FIG. 8A, in one example, one or more magnetic strips 125 are attached to a cuff 127 or other positioning system. The magnet strips 125 may be permanent magnets or activatable electromagnets. FIG. 8B depicts a cuff 127 with electromagnetic strips 129 powered by an external unit 131 through lead wires 133. The external unit 131 may also comprise a display 135 providing electrical current information to the electromagnetic field and/or data regarding detection of the ferromagnetic particles bound to the CMC of interest. Referring back to FIG. 8A, the cuff 127 or positioning system may be made from flexible material to allow close proximity between the magnets 125 and the attraction site 123. A securing assembly, such as a Velcro strip 137, may be used to secure the cuff 127 to the patient's body. As shown in FIG. 8B, the probe 100 with a magnetizable site 123 is positioned within the body and a magnetically labeled binding partner is introduced into the blood stream, body cavity or body lumen. The cuff is applied to the body at a location sufficient for the strips to generate a magnetic field at the attraction site and to draw the magnetically labeled binding partners.

The cuff or external securing system may optionally comprise one or more coils or loops that can act as a metal detector system to monitor the degree of concentrated antibody in the region of the cuff and probe. Metal detection systems are well known in the art and may be configured as very low frequency (VLF), pulse induction (PI) and beat-frequency oscillation (BFO) metal detection systems. In one embodiment, more sensitive metal detection configurations, such as VLF or PI, are preferred.

In another embodiment of the invention, depicted in FIG. 8D, the magnetizable probe 150 is configured so that it is capable of implantation within the body and does not require a permanent proximal attachment for manipulation and/or retrieval of the probe 150. A detachable or implantable probe 150 may be beneficial where detection of a CMC requires prolonged exposure to the body, but the probe 150 is not limited to this particular use. By detaching from its delivery tool, contact between the probe and the external surface of the body and the probe surface area within the body may be reduced. This may decrease the risk of thrombogenicity and/or infection created by the presence of the probe 150. Those with cancer or a history of cancer or other disease may be predisposed to clot formation and infection and may benefit from additional measures to reduce such risks.

In one embodiment, the probe 150 comprises a magnetizable zone and an engagement interface for reversibly engaging a delivery/retrieval tool. The magnetizable zone may comprise a magnetic coating on the probe 150. The engagement interface comprises a mechanical or friction interface capable of forming a mechanical or friction fit with a delivery/retrieval tool to facilitate implantation and removal of the probe. The engagement interface may be further configured to orient the probe with respect the delivery/retrieval tool to facilitate positioning and removal of the probe through narrow openings such as a blood vessel. The probe may optionally comprise an anchor system for maintaining the position of the probe in a general or particular location.

As shown in FIG. 8D, in one embodiment, the probe 150 has a stent-like configuration. The probe 150 may be self-expanding or balloon-expandable. One skilled in the art will understand that any of a variety of stent structures, configurations and materials may be used, including but not limited to nitinol, 316L stainless steel, platinum or platinum/iridium. The probe 150 may be dimensioned for placement in any of a variety of locations, including but not limited to cardiovascular system, a peripheral vein or artery, biliary system, urinary tract, gastrointestinal tract and other lumens or body cavities, natural or artificial. In one embodiment, the probe 150 has an average diameter of about 0.5 mm to about 2 mm. In another embodiment, the probe 150 has an average diameter of about 1 mm to about 8 mm. The probe 150 may have a length of about 5 mm to about 60 mm. In another embodiment, the probe 150 a length of about 10 mm to about 30 mm.

Stent retrieval is known in the art and may be performed in several ways. Representative patents include but are not limited to U.S. Pat. No. 6,569,181 to Burns and U.S. Pat. No. 6,187,016 to Hedges et al., herein incorporated in their entirety by reference. The stent support may further comprise one or more engagement elements to facilitate retrieval of the stent from the body by a retrieval tool.

In another embodiment of the invention, a fiberoptic detection system is provided to detect bound CMC. Referring to FIG. 9, the probe 100 comprises a probe body 126 having a proximal end 128 and a distal end 130. A polymer gel cavity 108 is located on the probe body 126, typically about the distal end 130 but other locations may also be used. Two or more optical fibers 132, 134 are located between the proximal end 128 of the probe body 126 and the polymer gel cavity 108. At least one fiber is an illumination fiber 132 for providing light to the polymer cavity. At least one other fiber is a detection fiber 134 used to analyze the light found in the polymer cavity. Optical fibers are well known in the art and often comprise glass or plastic fibers made from low OH silica, quartz or nylon. The fibers may be single or multi-mode fibers and have a diameter of about 2 microns to about 200 microns. The fibers may or may not include a polyimide or Teflon jacket. Additional insulation materials 136 or tubing may be used about the optical fibers for additional protection or to block stray light. The insulation 136 can be a polymer material, including but not limited to PTFE, polyurethane, nylon or polyimide. In one embodiment, a molded, adhesive back-filled or separate tube with a diameter of about 250 microns may be used. The proximal ends 138, 140 of the optic fibers 132, 134 terminate at one or more fiberoptic connectors 142. Standard SMA connectors may be used, but other standard or proprietary connectors may be substituted. The connectors are configured to plug into a monitoring system 144 comprising an illumination lamp to provide a light source for the illumination fiber 114, a detection system for analyzing the light spectrum within the cavity, and a display monitor for displaying the providing information regarding the probe. One skilled in the art can configure each of these and other components of the detection system for a particular purpose.

To detect the binding partner(s) attracted or collected by the probe, any of a variety of optically detectable components may be linked to the binding partner(s). In one embodiment, the binding partner comprises a fluorescent dye is attached to an antibody or other type of binding partner. Fluorescent dye labeled antibodies are well known in the art.

In another embodiment, the binding partner for the CMC of interest is optionally linked to a quantum dot. Quantum dots are small crystals with a particle size of about 10 nanometers or more that flow when they are stimulated by ultraviolet light. The wavelength, or color, of the light depends on the size of the crystal. Latex beads filled with these crystals can be designed to bind to specific DNA sequences. By combining different size quantum dots within a single bead, the quantum dot binding partners can release distinct colors and intensities of light by the detection fiber of the fiberoptic probe.

FIGS. 10A and 10B depict another embodiment of the invention comprising a fiber optic detection system, wherein a microporous membrane or layer 146 or coating is utilized, rather than a polymer gel cavity. The porous membrane 146 may be a polymeric, metallic or ceramic, but is preferably a porous polymer gel such as a silicone or polyurethane. The polymer layer 146 is at least about 2 mm to about 6 mm in length with a thickness of about 2 microns to about 100 microns and a porosity of about 10% to about 60%. The polymer used generally has a hardness of about 30 A to about 60 A.

In one embodiment, a method for using a fiberoptic probe in a patient is provided. The probe is inserted into the vasculature of the patient. Antibody linked to a quenching agent is injected into the vasculature and allowed to bind to a CMC. Binding of a CMC to the antibody causes a shift in the energy spectrum of the quenching agent, thereby causing a detectable wavelength shift when exposed to light from the illumination fiber of the probe. The detection fiber is able to sense the wavelength shift and transmit the optical information the display and detection system.

In other embodiments, a combined fiberoptic and impedance system detection system is provided in the probe to detect more than one CMC or to enhance the reliability of the detection scheme.

In one embodiments of the invention, the probe further comprises additional sensor assemblies to detect other characteristics of the probe environment or CMC. Information from these other sensor assemblies may be used to further refine the CMC collection or detection or to provide complementary information. These other sensor assemblies may include but are not limited to a temperature probe, pH sensor or fiberoptic sensor for assessing other factors such as glucose or potassium levels. A fluid reservoir may be provided adjacent to the microporous structure to further assist with CMC detection. The reservoir may contain certain chemicals, enzymes or other agents that are part of these other sensor assemblies. Examples include but are not limited to a pH sensitive gel or fluid.

Other molecules or components may be bound to the probe to facilitate or support the function of the attraction structure. In one embodiment, heparin is bound to the probe to resist thrombus formation that may affect the function of the attraction structure with extended exposure time to the body. Heparin coating of medical devices is well known in the art, as described by Hsu et al. in U.S. Pat. No. 5,417,969, herein incorporated in its entirety by reference. In another embodiment, a streptokinase coating is provided to resist clot formation (Niku S D et al., Isolation of lymphocytes from clotted blood, J Immunol Methods. 1987 December 4;105(1):9-14, herein incorporated by reference). Other materials that may be bonded to the probe include but are not limited to hydrogels or other lubricious coatings, as described by Hostettler et al. in U.S. Pat. No. 5,919,570, and antimicrobial agents, as described by Raad and Sherertz in U.S. Pat. No. 5,688,516, herein incorporated in their entirety by reference. An antimicrobial component may reduce the risk of probe colonization by infectious bacterial and fungal organisms for a probe placed into a body for an extended period of time. Such antimicrobial agents may include but are not limited to aminoglycoside, amphotericin B, ampicillin, carbenicillin, cefazolin, cephalosporin, chloramphenicol, clindamycin, erythromycin, gentamicin, griseofulvin, kanamycin, methicillin, nafcillin, novobiocin, penicillin, polymyxin, rifampin, streptomycin, sulfamethoxazole, sulfonamide, tetracycline, trimethoprim, and vancomycin.

The probe may further comprise an optional elution zone capable of retaining and releasing one or more substances such as drug compounds, reagents or other substances. In one embodiment, the elution zone releases a substance that enhances release of a CMC from the body. In another embodiment, the elution zone releases a substance that facilitates detection of a CMC, including but not limited to Ab labeled fluorescent dyes. In still another embodiment, the elution zone releases a substance capable of reducing a body's immune response to an antigenic element on the probe. In another embodiment, the elution zone is capable of releasing one or more treatment agents for reducing fibrin deposition onto the binding zone and other portions of the probe. Fibrin deposition may decrease or affect the binding of CMCs to their binding partners into the binding zone. Agents that may be released from the elution zone include but are not limited to dexamethasone, paclitaxel, unfractionated heparin, low-molecular weight heparin, enoxaprin, synthetic polysaccharides, ticlopinin, dipyridamole, clopidogrel, fondaparinux, streptokinase, urokinase, r-urokinase, r-prourokinase, rt-PA, APSAC, TNK-rt-PA, reteplase, alteplase, monteplase, lanoplase, pamiteplase, staphylokinase, abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban, bivalirudin, and pentoxifylline.

D. Insertion and Placement of Collection Probe

The collection probe may be inserted in a variety of ways and to a variety of locations within the body. In some situations, the probe may be inserted during a cancer surgery where access to sentinel sites of disease recurrence is readily accessible. For instance, following a mastectomy and axillary node dissection for breast cancer, a collecting probe may be implanted during the same procedure into the lymphatic ducts draining the breast. Such as site may provide earlier detection of recurring disease and may also increase the yield from such surveillance. Similarly, placement of the collection probe surgically may also allow or subcutaneous implantation into a large vein while the patient is still under anesthesia, thereby decreasing the risk of infecting the device compared to percutaneous insertion.

The device may also be configured for percutaneous insertion. Some embodiments of the device allow insertion of the probe into existing long-term access sites such as a Hickman catheter, Portacath, or a peripherally inserted central catheter (PICC) line or variants thereof. Similarly, the probe may also be configured for insertion through central venous catheters inserted into the femoral or jugular vein, or large-bore IV access site. For example, a Portacath is an implantable venous access device that is frequently used in cancer patients to provide long-term vascular access for chemotherapy. A detection probe placed into a Portacath or a Portacath variant may serve a dual function of treating the cancer and provide the ability to monitor treatment effect.

In use, a probe having at least one binding partner is provided. The probe is advanced to a site where a binding zone on the probe will be exposed to a carrier such as blood which may periodically contain a marker of interest. The probe is left in place for an evaluation period, to allow the marker to become bound to the binding partner. The probe is thereafter withdrawn, and evaluated to determine the presence of any marker carried by the binding zone.

In one application, the probe is advanced through an access tube to position the binding zone at an intralumenal site within an artery or vein. The binding zone is left at the site for an evaluation period of generally at least about one hour, in come applications at least about four or six hours, and for certain markers at least about 12 hours or 24 hours or more. This allows collection of at least a first quantity of a target marker from a first release of marker into the blood, and in certain applications at least also a second quantity of the target marker from a second release of marker into the blood, the first and second releases separated in time from each other. The first and second quantities of the target marker may be collected on the same probe. Alternatively, during the evaluation period, a first probe may be withdrawn from the site and replaced by at least a second probe, which carries the same or a second binding partner.

The device may be inserted through any of a variety of access methods known to interventional radiology, cardiology, gastroenterology and other medical and veterinary disciplines. These procedures may include but are not limited to endoscopic retrograde cholangiopancreatography (ERCP) for placement into the biliary tree or pancreas, transseptal puncture for placement into the arterial portion of the cardiovascular system, lumbar puncture into the cerebrospinal fluid, and cystoscopy for placement into the urinary tract.

In some embodiments of the invention, the proximal end of the probe has a closed end without an electrical or fiberoptic connector. The proximal ends of the lead wires or fiberoptic lines terminate in a receiving and storage assembly that is capable of receiving the impedance or optical information detected by the probe and to store the data for retrieval at a later date. In a further embodiment of the invention, the receiving and storage assembly further comprises a wireless transmitter for transmitting the data to a remote base. Wireless transmission is well known in the art. For example, a Bluetooth wireless system may be used to transmit data from the probe to a computer or remote base.

E. Ex Vivo Probe Assessment

The capture of nucleic acids from an in vivo device can be monitored ex vivo using standard qualitative and quantitative molecular assays. The assays can directly measure the nucleic acids or amplify them to measure them. The assays can be probe-, sequence-or affinity ligand-based. The assessment of DNA/RNA in body fluids ex vivo is known and currently available. These include but are not limited to gel electrophoresis, real time quantitative polymerase chain reaction (PCR), probe based chromatographic assays. For DNA analysis, this will include microsatellite analysis for loss of heterozygosity or by single nucleotide polymorphism (SNP). Other DNA markers can include mutations, amplifications, insertions and translocations. This may be specific or multiple sites of the DNA from tumor cells. RNA analysis will involve assessment of mRNA of transcripts of specific genes related to the tumor cells. The mRNA transcript may be of the whole or part of the full transcript or a truncated derivative of the transcript. The procedure may also include chromatin and DNA complexes related to specific genomic regions of tumor cells. The procedure may also include assessment of acetylated and de-acetylated or modified regions of the chromatin and histones surrounding a specific gene. The procedure may also include assessment of methylation or demethylation of gene promoter regions.

As mentioned previously, separating and/or distinguishing a bound binding partner from an unbound binding partner on the probe is known in the art. In some embodiments of the invention, the unbound antibody is separated from the probe while the CMC-bound antibody remains on the probe. An impedance measurement system may then be used to analyze the presence of remaining bound antibody on the probe.

Assessment of antibody or protein-based markers is currently available and may include but is not limited to affinity binding assays, mass spectroscopy, and ELISA. Similarly, carbohydrate markers are also known and may include affinity or ligand-based capture assays and mass spectroscopy. One skilled in the art can select one or more assays based upon the particular marker or markers of interest.

One embodiment of the invention comprises a percutaneously insertable device used with injectable antibodies that recognize tumor-related cell surface proteins/glycoproteins (i.e.: cMet, HER2/neu, beta-Human chorionic gonadotropin (HCG), MUC-1, etc) or glycolipids (gangliosides GM2, GD2). The antibodies can capture and bind the circulating tumor cells in the blood or body fluid. Single or multiple antibodies to a specific cell surface marker or multiple markers may be used. The attraction device or catheter with the attracted tumor cells can be removed and subjected to standard ex vivo isolation methods known in the art for RNA, DNA, carbohydrate and protein isolation and purification. The isolation of these cell products is one approach to identify their specificity. Another approach is to isolate the cells and assess them as whole cells. These approaches are advantageous in providing a unique in vivo enrichment method for the collection of circulating tumor cells and their subcomponents, such as DNA, RNA and proteins, for further evaluation and assessment.

In some embodiments, the cells can be separated from the device by turning off the active attraction force created by an electromagnetic attraction site on the device. Once separated from the device, the cells can undergo respective component isolation. In other embodiments, the cells are analyzed while still attached to the device. In one example, cells can be processed, purified and quantitated for specific nucleic acids such as RNA and DNA by methods known in the art. To assess the amount of nucleic acids, one can perform qualitative and/or quantitative analysis for specific RNA and DNA markers that are tumor-related. These markers may be different from the antibody specific markers that are used to capture the cells. The antibody used to capture markers may also be used.

In one embodiment, cells are captured using antibody to c-Met, then assessment for cMet mRNA expression in the cells is performed qualitatively or quantitatively by realtime PCR. PCR provides amplification of the target mRNA marker and allows for detection through many available approaches including but not limited to as gel electrophoresis, realtime PCR thermocyclers, etc.

In one embodiment, tumor mRNA markers for assessment can include markers most prevalent in the type of cancer being assessed. For example, in melanoma one could assess for MART-1 mRNA. For breast cancer one can assess mammoglobin. Less prevalent markers may also be used. Quantitative marker detection may be used to rule out false positives. This provides another layer of specificity to the detection scheme. Also, to increase the sensitivity of the detection scheme, multiple markers can be used to assess for isolated tumor cells. One can also assess for specific DNA markers such as mutations, loss of heterozygosity, amplification, translocation, etc. Specific genetic changes may be related to specific cancers or groups of cancers. Specific genetic changes can be used in combination with multiple marker detection approaches. Some examples include detection of BRAF mutation at V600 for melanoma, methylation of RASSF1a promoter site, or LOH at 9p21. The use of specific nucleic markers can be used to determine specific types of cancers, level of disease malignancy, disease aggressiveness, prognostic and predictive values and other information.

In one approach, proteins are isolated and purified by direct isolation. These proteins can be assessed by ELISA for specific tumor markers, Western Blot approaches, mass spectrometry, protein arrays, ProteinChips, antibody based assays, affinity protein based assays, etc in a quantitative and qualitative manner. The approaches can be used for glycoproteins and other carbohydrate markers. The use of specific protein/glycoprotein/carbohydrate markers can be used to determine specific types of cancers, level of disease malignancy, disease aggressiveness, prognostic and predictive values and other information.

Another approach is to elute the cells. Cells attracted to the catheter can be evaluated using conventional histopathologic and immunocytochemical staining methods that characterize the collected cells of interest. These cells can be evaluated directly on the catheter or, in one embodiment, the cells are separated from the catheter by inactivating the attraction force. Standard methods to disrupt tumor cell complementary antibody binding may then be used to separate, for example, the antibody/magnetic particle combination from the tumor cell. Current methods include mechanical separation (such as scraping and/or washings with saline, buffered solutions, or media), chemical dissociation techniques such as washing the catheter/antibody/tumor cell complex with pH buffered solutions (such as PBS with EDTA or salts that disrupt antibody binding to cells but not destroy the cells, etc.), thus allowing the cells to be collected intact after separation from the antibody and assessed by conventional methods such as immunostaining procedures. In still another embodiment, cells may also be released by disrupting the antibody-cell complex from the device. After isolation, the cells can be immunostained with specific antibodies against tumor cell surface markers or intracellular markers. The assessment of tumor cells may be performed by conventional immunopathology for tumor cell diagnosis, but other approaches are known in the art, including but no limited to immunostained cells by FACs analysis. In these approaches, multiple antibodies can be used for detection to improve sensitivity and specificity for specific cells. Also, some approaches allow detection of the number of cells detected for quantitation of disease level. Cells can be also assessed by conventional or non-conventional stains and dyes that are not antibody-based. Still another approach is in situ hybridization with nucleic acids or derivative molecules that are complimentary. The above approaches for detection of eluted cells, intact or not intact, for specific components (protein, nucleic acids, etc) can be approached quantitatively or qualitatively. The approaches can be by individual or combination of methods.

While this invention has been particularly shown and described with references to embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention. For all of the embodiments described above, the steps of the methods need not be performed sequentially. 

1. A method for detecting disease, comprising the steps of: providing a binding agent attraction device; inserting the device into a body; introducing a binding agent into the body by injecting the binding agent into the bloodstream; attracting at least a portion of the binding agent to the attraction device; and assessing the binding agent attracted to the attraction device.
 2. The method of claim 1, wherein the assessing step is performed by impedance-based detection of the binding agent.
 3. The method of claim 1, wherein the assessing step is performed by optical detection of the binding agent.
 4. A method for detecting disease, comprising the steps of: providing a binding agent attraction device; inserting the device into a body; introducing a binding agent into the body by eluting the binding agent from an implant within the body; attracting at least a portion of the binding agent to the attraction device; and assessing the binding agent attracted to the attraction device.
 5. The method of claim 4, wherein the assessing step is performed by impedance-based detection of the binding agent.
 6. The method of claim 4, wherein the assessing step is performed by optical detection of the binding agent.
 7. A method for detecting disease, comprising the steps of: providing a binding agent attraction device; inserting the device into a body; introducing a binding agent into the body by ingestion of the binding agent into the body; attracting at least a portion of the binding agent to the attraction device; and assessing the binding agent attracted to the attraction device.
 8. The method of claim 7, wherein the assessing step is performed by impedance-based detection of the binding agent.
 9. The method of claim 7, wherein the assessing step is performed by optical detection of the binding agent.
 10. A method for detecting disease, comprising the steps of: providing a binding agent attraction device; inserting the device into a body; introducing a binding agent into the body; attracting at least a portion of the binding agent to the attraction device; and assessing the binding agent attracted to the attraction device by impedance-based detection of the binding agent.
 11. The method of claim 10, wherein the introducing step is performed by injecting the binding agent into the bloodstream.
 12. The method of claim 10, wherein the introducing step is performed by eluting the binding agent from an implant within the body.
 13. The method of claim 10, wherein the introducing step is performed by ingestion of the binding agent into the body.
 14. A method for detecting disease, comprising the steps of: providing a binding agent attraction device; inserting the device into a body; introducing a binding agent into the body; attracting at least a portion of the binding agent to the attraction device; and assessing the binding agent attracted to the attraction device by optical detection of the binding agent.
 15. The method of claim 14, wherein the introducing step is performed by injecting the binding agent into the bloodstream.
 16. The method of claim 14, wherein the introducing step is performed by eluting the binding agent from an implant within the body.
 17. The method of claim 14, wherein the introducing step is performed by ingestion of the binding agent into the body. 